The lipooligosaccharides of Haemophilus influenzae are important virulence factors. The lipid A portion of the lipooligosaccharide (LOS) is responsible in part for the toxicity associated with this organism. The role of the oligosaccharide portion of the LOS in pathogenesis of H. influenzae infection is less clear. Our preliminary studies outlined in the progress report have shown that these oligosaccharides from Hib and NTHi contain structures which mimic at least three different human surface glycolipid antigens. In addition, we have cloned two distinct clusters of biosynthesis genes associated with expression of these oligosaccharides. A 7.2 kb portion of one of these clusters has been sequenced and deletion or site directed H. influenzae mutants generated. Transformation of this modified DNA into Hib strain A2 has resulted in isogenic Hib strains with stable LOS mutations which are less invasive in human tissue culture cell lines than the isogenic wild type Hib strain. Our studies have resulted in physicochemical analysis of the structure of oligosaccharides from Hib and NTHi strains. This analysis has shown a unique deep core structure for the H. influenzae LOS oligosaccharide. Simultaneous substitution of the branched tri-heptose in this structure can lead to synthesis of a complex array of oligosaccharide side chains. In addition, physicochemical studies have provided structural confirmation of the oligosaccharide modifications produced by one of the oligosaccharide biosynthesis clusters we have identified. Our hypothesis in this resubmission states that H. influenzae LOS oligosaccharides play a role in pathogenesis by immune evasion through molecular mimicry and act to promote close range adherence to human cells and possibly facilitate human cell invasion. To resolve this hypothesis, we plan to continue to define the nature of the process involved in the biosynthesis of the Hib and NTHi oligosaccharides. In addition, we propose the study of LOS from a H. influenzae biogroup aegyptius (HIA) strain which has been shown to be invasive for the human nasopharyngeal organ culture model. By generation of Hib, NTHi and HIA mutants expressing a limited repertoire of oligosaccharides, we will define the oligosaccharide residues responsible for adherence and invasion of human cells. Finally, we will complete the structural analysis of the LOS oligosaccharide of Hib strain A2 and NTHi strain 2019 and institute analysis of the H. influenzae biogroup aegyptius LOS. These goals will be accomplished by the following Specific Aims: 1) Characterization of H. influenzae LOS oligosaccharide biosynthesis gene clusters. 2) Physiochemical characterization of the oligosaccharide portion of Hib, NTHi and HIA LOS. 3) Definition of the LOS oligosaccharide structure(s) involved in adherence and invasion in tissue culture and organ culture systems. To carry out the structural aspects of these specific aims, we will be using NMR and mass spectrometry techniques. Specifically, we will employ electrospray ionization and matrix-assisted laser desorption mass spectrometry to assess structural heterogeneity of LOS. Tandem MS/MS will then be used either on a four sector instrument or through low energy ESI MS/MS to determine specific oligosaccharide structures in these LOS species.